The carboxypeptidase family of exopeptidases constitute a diverse group of enzymes that hydrolyze carboxyl-terminal amide bonds in polypeptides. Carboxypeptidases from grains such as wheat and barley have been isolated and sequenced (Baulcombe et al., J. Biol. Chem., 262: 13726-13735 [1987]; Svendsen and Breddam, Carlsberg Res. Commun., 52: 285-295 [1987]), as well as carboxypeptidases from bacteria, and a carboxypeptidase Y has been isolated from yeast vacuoles and sequenced. Valls et al., cell, 48:887-897 [1987]; Svendsen et al., Carlsberg Res. Commun., 47: 15-27 [1982]). The sequences of carboxypeptidase B from crayfish (Titani et al., Biochemistry, 23: 1245-1250 [1984]) and African lungfish (Reeck and Neurath, Biochemistry, 11: 3947-3955 [1972]) have been determined. A large number of mammalian tissues also produce these enzymes.
The exocrine pancreas synthesizes and secretes a subset of zinc metalloproteases. Two members of this metalloprotease family, carboxypeptidase A and carboxypeptidase B, have been purified from bovine pancreas and characterized. Barrett and MacDonald, Mammalian Proteases, a Glossary and Bibliography, Vol. 2 (Academic Press, London 1985), and references cited therein, including Titani et al., Proc. Nat. Acad. Sci. USA, 72: 1666-1670 [1975]; Bradshaw et al., Biochemistry, 10: 961-972 [1971]; Wade et al., Biochimie, 70: 1137-1142 [1988]. Bovine carboxypeptidase B was found to inhibit the activation of plasminogen by t-PA in the presence of degraded fibrin. Pannell et al., J. Clin. Inv., 81: 853-859 (1988).
The bovine carboxypeptidases A and B have similar amino acid sequence, three-dimensional structure, and catalytic mechanisms, but differ in the substrate upon which they act. Carboxypeptidase A hydrolyzes carboxyl-terminal amide bonds in which the adjoining carboxy-terminal amino acid contains an aromatic or branched aliphatic side chain, whereas carboxypeptidase B prefers Lys or Arg residues as substrates at the carboxyl terminus.
Three carboxypeptidases differing in their substrate specificity (designated CPA1, CPA2, and CPB) were isolated from rat pancreas. Gardell et al., J. Biol. Chem., 263: 17828-17836 (1988). The genes for rat CPA1 and CPA2 have been cloned and sequenced. Gardell et al., supra; Clauser et al., J. Biol. Chem., 263: 17837-17845 (1988). See also Quinto et al., Proc. Natl. Acad. Sci. USA, 79: 31-35 (1982). Sequences have also been obtained of fragments from porcine carboxypeptidase A (Vendrell et al., Biochem. Biophys. Res. Commun., 141: 517-523 [1986]) and carboxypeptidase B (Aviles et al., Biochem. Biophys. Res. Commun., 130: 97-103 [1985]).
In addition to the pancreas, mast cells also contain large amounts of carboxypeptidase A, including rat and mouse peritoneal connective tissue mast cells [Everitt and Neurath, FEBS Lett., 110: 292-296 (1980); Schwartz et al., J. Immunol., 128: 1128-1133 (1982); Serafin et al., J. Immunol., 139: 3771-3776 (1987)], mouse Kirsten sarcoma virus-immortalized mast cells [Reynolds et al., J. Biol. Chem., 263: 12783-12791 (1988)], and human skin mast cells [Goldstein et al., J. Immunol., 139: 2724-2729 (1987); Goldstein et al., J. Clin. Invest., 83: 1630-1636 (1989)].
Mast cell carboxypeptidase A is a neutral to basically charged protein stored in the secretory granules of rat and mouse peritoneal connective tissue mast cells and mouse interleukin-3-dependent bone marrow-derived mast cells as a fully active enzyme bound ionically to acidically charged proteoglycans. Basically charged serine endopeptidases are similarly stored. The close approximation of carboxypeptidase A and serine protease activities within the protease-proteoglycan macromolecular complex is thought to facilitate sequential endopeptidase and exopeptidase cleavages of common protein substrates. Kokkonen and Kovanen, J. Biol. Chem., 264: 10749-10755 (1989); Kokkonen et al., J. Biol. Chem., 261: 16067-16072 (1986).
Mast cell carboxypeptidase A has been isolated from the secretory granules of mouse peritoneal connective tissue mast cells and from a mouse Kirsten sarcoma virus-immortalized mast cell line, and a cDNA encoding this exopeptidase has been cloned. Reynolds et al., J. Biol. Chem., 264: 20094-20099 (1989). In addition, the mast cell carboxypeptidase A from humans has been cloned and its sequence determined. Reynolds et al., Proc. Natl. Acad. Sci. USA, 86: 9480-9484 (1989).
Other mammalian carboxypeptidases besides carboxypeptidase B that specifically remove terminal basic amino acids include carboxypeptidase H (also known as enkephalin convertase or carboxypeptidase E), carboxypeptidase M, and carboxypeptidase N. The mammalian arginine/lysine carboxypeptidases have important functions in many biological processes, including protein digestion, activation, inactivation, or modulation of peptide hormone activity, and alteration of the physical properties of proteins and enzymes.
The actual role of these carboxypeptidases in vivo is likely related to their localization as well as their physical properties. For example, pancreatic carboxypeptidase B is not normally found outside the pancreas or small intestine except in the case of acute pancreatitis, consistent with its major function in protein degradation in the digestive tract. Delk et al., Clin. Chem., 31: 1294-1300 (1985).
In contrast, human plasma carboxypeptidase N circulates in plasma as a large tetrameric complex of two active subunits (48-55 kD) and two glycosylated inactive subunits (83 kD) that stabilize the active subunits and keep them in the circulation. Carboxypeptidase N protects the body from potent vasoactive and inflammatory peptides containing COOH-terminal Arg or Lys released into the circulation. Erdos (ed.) in Handbook of Experimental Pharmacology, Vol. 25, Supplement, pp. 428-487, (Springer-Verlag, Heidelberg, 1979); Plummer and Hurwitz, J. Biol., Chem., 253: 39-7-3912 (1978). Recently, carboxypeptidase N has been cloned and sequenced. Tan et al., J. Biol, Chem., 265: 13-19 (1990); Gebhard et al., Eur. J. Biol. Chem., 603-607 (1989); Skidgel et al., Biochem. Biophys. Res. Commun., 154: 1323-1329 (1988).
Carboxypeptidase E (or H), an arginine/lysine carboxypeptidase with an acid pH optimum, is located in secretory granules of pancreatic islets, adrenal gland, pituitary, and brain. Zuhlke et al., Ciba Found. Symp., 41: 183-195 (1975); Davidson and Hutton, Biochem. J., 245: 575-582 (1987); Hook and Loh, Proc. Natl. Acad. Sci. USA, 81: 2776-2780 (1984). It is believed that this enzyme removes the residual COOH-terminal Arg or Lys remaining after initial endoprotease cleavage during prohormone processing at the intragranular acid pH. Fricker, Annu. Rev. Physiol., 50:309-321 (1988). Carboxypeptidase E has been isolated, cloned, and sequenced from different sources (rat: Frickler et al., J. Mol. Endocrinol., 3: 666-673 [1989]; bovine: Fricker et al., Nature, 323: 461-464 [1986]; human: Hook and Affolter, FEBS Lett., 238: 338-342 [1988]; Manser et al., Biochem. J., 267: 517-525 [1990]).
Carboxypeptidase M is a membrane-bound arginine/lysine carboxypeptidase found in many tissues and cultured cells. Skidgel, Trends Pharmacol. Sci., 9: 299-304 (1988). Recently, it has been purified to homogeneity from human placenta. Skidgel et al., J. Biol. Chem., 264: 2236-2241 (1989). Because of its presence on plasma membranes and optimal activity at neutral pH, it may act on peptide hormones at local tissue sites where it could control their activity before or after interaction with specific plasma membrane receptors. Sequencing has shown carboxypeptidase M to be a unique enzyme that exhibits some similarity to carboxypeptidases A, B, E, and N. Tan et al., J. Biol. Chem., 264: 13165-13170 (1989).
It is an object of the present invention to identify a novel carboxypeptidase B isolated from plasma that shares some common structural features and catalytic and substrate binding sites with carboxypeptidase A and with pancreas carboxypeptidase B, and in its human embodiment shares the most sequence identity with known carboxypeptidases A and B.
It is another object to provide nucleic acid encoding such a polypeptide and to use this nucleic acid to produce the polypeptide in recombinant cell culture for diagnostic use or for potential therapeutic use in hemostatic regulation.
It is yet another object to provide derivatives and modified forms of such a new polypeptide, including amino acid sequence variants and covalent derivatives thereof.
It is an additional object to prepare immunogens for raising antibodies against such new polypeptide, as well as to obtain antibodies capable of binding it.
These and other objects of the invention will be apparent to the ordinary artisan upon consideration of the specification as a whole.